1a.Objectives (from AD-416)
1. To develop ultra sensitive Taq-Man PCR diagnostic system for Improve detection of Ca. Liberibacter asiaticus.
2. To design a Ca. Liberibacter genus-specific real-time PCR technology for general detection of Candidatus Liberibacter species.
1b.Approach (from AD-416)
1. Cloning, sequencing and characterizing genomic sequences of citrus Candidatus Liberibacter species using novel molecular technique.
2. Design and validate sensitivity and specificity of diagnostic systems.
This Trust Agreement is in support of Objective 1.B of the parent project. The goal of the project is to develop improved diagnostic systems for reliable and sensitive detection of ‘Candidatus Liberibacter’ (CL) bacterial species associated with citrus huanglongbing. Two novel surveillance systems for CL species detection were developed and are currently under evaluation. The first system is called “single tube dual primer Taq-Man polymerase chain reaction (PCR)". This detection system has improved sensitivity as compared with standard Taq-Man PCR. The whole process is completed in a single closed tube, thus eliminating the potential risk of cross-contamination commonly associated with conventional two-tube PCR. The second system is a fluorescent dye-based Real-Time PCR system which employs a pair of CL universal primers which can amplify all four known CL species; “CL asiaticus”, “CL africanus”, “CL americanus” and “CL solanacearum”. The amplification products have thermo-stability characteristics unique for each bacterial species and that can be reliably distinguished by high resolution melting curve analyses. Both systems are robust and cost-effective for reliable detection, quantitation and identification of CL species in plants and insects. Both systems provide high throughput capabilities suitable for large scale, year around quarantine screening and epidemiological studies.